This is the U.S. National Phase under 35 U.S.C. 371 of International Application PCT/EP98/02830, filed May 7, 1998.
The present invention relates to a polynucleotide, in particular a cDNA, which codes for L-galactono-xcex3-lactone dehydrogenase (GLDase), an enzyme involved in the biosynthesis of ascorbic acid (vitamin C) in plants. The invention further relates to the use of this cDNA for the synthesis of the enzyme and for the production of transgenic plant and animal cells, plant tissues and plants producing the enzyme.
Ascorbic acid is synthesized in all higher plants and in almost all higher animals, with the exception of humans and other primates, the guinea pig and a number of birds. Opinions differ concerning the presence of ascorbic acid in micro-organisms. It appears to be present in a number of yeasts, although there are also reports which suggest that ascorbic acid analogues are found in micro-organisms.
In the animal and plant kingdom, ascorbic acid is formed by different routes. In animals, glucose is the primary precursor for the biosynthesis of ascorbic acid, and the last step in the biosynthetic pathway is catalyzed by a microsomal enzyme: L-gulono-xcex3-lactone oxidase. This enzyme has already been isolated from rat, goat and chicken liver and kidney tissues.
The pathway of ascorbic acid biosynthesis in plants, however, is not yet entirely clear, but there are indications that at least two different biosynthetic pathways exist. Isherwood et al., Biochem. J. 56:1-15 (1954) postulated that the biosynthesis of ascorbic acid starting from D-galactose proceeds via L-galactono-xcex3-lactone to L-ascorbic acid. Mapson et al., Biochem. J. 56:21-28 (1954) were the first to study this oxidation of L-galactono-xcex3-lactone to ascorbic acid, a reaction which is catalyzed by L-galactono-xcex3-lactone dehydrogenase.
The presence of L-galactono-xcex3-lactone dehydrogenase activity has been described for different plants, including pea, cabbage and potato. Ôba et al., J. Biochem. 117:120-124 (1995) have recently purified the enzyme activity from sweet potato tubers.
Distinct from this biosynthetic pathway, however, an alternative pathway has been proposed which takes as starting point the conversion of D-glucose, and proceeds via L-glucosone and L-sorbosone to ascorbic acid. An NADP-dependent dehydrogenase, which catalyses the conversion of L-sorbosone to ascorbic acid, has been partially purified from bean and spinach leaves (Loewees et al., Plant Physiol. 94:1492-1495 (1990)).
The primary function of ascorbate is as a reducing agent. This is universal. Ascorbic acid is also important as a cofactor for certain enzymatic reactions, including the production of collagen in vertebrates. Since humans are completely dependent on ingested food for the acquisition of ascorbate, it is desirable to increase the vitamin C content of plants and fruit.
Owing to its reducing activity, vitamin C plays a role in the protection of plants and animals against environmental stresses including heat, cold, drought, oxidative stress etcetera. Less stress-sensitive or even stress-resistant plants can therefore play an important part in the economy and agriculture of the world.
It is the object of the present invention to create the possibility of genetically modifying plants such that they contain an increased content of ascorbic acid relative to non-modified plants.
For this purpose the invention provides a polynucleotide in isolated form, which polynucleotide codes for a protein with the activity of the enzyme L-galactono-xcex3-lactone dehydrogenase, which polynucleotide comprises at least the L-galactono-xcex3-lactone dehydrogenase activity-determining parts of the coding part of the nucleotide sequence, which is shown in FIG. 3, or a sequence derived therefrom on the basis of the degeneration of the genetic code. The invention is of course not limited to polynucleotides with exactly the same sequence as that shown in FIG. 3. It will be apparent to the molecular biologist skilled in the techniques that a certain degree of modification of the sequence shown in FIG. 3 is permitted while still falling within the scope of the claim. The polynucleotide is for instance the cDNA shown in FIG. 3.
Polynucleotides according to the invention can be used in the production of transgenic plant and animal cells, plant tissues or plants with an increased content of the enzyme L-galactono-xcex3-lactone dehydrogenase relative to non-transgenic plant cells, plant tissues or plants. Such an increased concentration of GLDase will result in plant cells, plant tissues or plants with an increased content of ascorbic acid and with an increased capacity for biosynthesis relative to non-transgenic plant cells, plant tissues or plants.
Plants which can advantageously be used for transformation with the polynucleotide according to the invention are for instance thale cress (Arabidopsis thaliana), tobacco (Nicotiana tabacum), tomato, potato, or corn, without this list being limitative.
Polynucleotides according to the invention can likewise be expressed in eukaryotic cells, such as yeast cells or mammalian cells, in particular fibrosarcoma cells.
The invention further relates to a recombinant L-galactono-xcex3-lactone dehydrogenase which can be obtained by expression of a polynucleotide according to the invention in a suitable host. The recombinant L-galactono-xcex3-lactone dehydrogenase can be isolated from transgenic plant tissues or transgenic plants, but also from yeasts or from animal cells.
The invention also relates to a transformation system, comprising a transformation vector or set of vectors, at least one of which includes a nucleotide sequence which codes for the enzyme L-galactono-xcex3-lactone dehydrogenase The transformation system preferably comprises Agrobacterium and a binary vector.
Plants or plant tissues with an increased ascorbic acid content can be produced by transforming a plant cell with a gene construct comprising at least the polynucleotide specified in the invention, optionally linked to targeting sequences for specific organelles, and/or in the presence of suitable transcription and/or translation regulation factors, and regenerating from the plant cell a transgenic plant or plant tissue. The gene construct with the polynucleotide according to the invention can optionally be combined with other genes coding for enzymes which can interfere in the ascorbic acid synthesis, such as L-sorbosone dehydrogenase, UDP-glucuronic acid epimerase, D-galacturonic acid dehydrogenase and ascorbate-regulating enzymes, which may determine the rate of ascorbic acid synthesis
The enzyme may ultimately be targeted to a particular part of the plant cell, such as the cytoplasm, vacuoles, chloroplasts, mitochondria, lysosomes, endoplasmatic reticulum, Golgi apparatus.
Eukaryotic cells expressing the enzyme GLDase can be obtained by transfection with the polynucleotide according to the invention.
Finally, the invention relates to a new method for purifying the enzyme L-galactono-xcex3-lactone dehydrogenase. This method comprises of passing a protein extract of cauliflower florets through an ion exchange column: collecting a number fractions eluting from the column and determining the GLDase activity of the fractions; combining fractions with GLDase activity and passing thereof through a Phenyl Sepharose CL 4B column; collecting the column eluate in a number of fractions and determining the GLDase activity of the fractions; combining those fractions with GLDase activity and passing thereof through a gel filtration column; collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions; combining the fractions with GLDase activity and passing through an FPLC Resource Q-column; collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions; combining the fractions with GLDase activity and passing thereof over an FPLC Poros 20 SP-column; collecting a number of fractions eluting from the column and determining the GLDase activity of the fractions. The enzyme purified by us is lycorine-insensitive, in contrast to the literature which states that L-galactono-xcex3-lactone dehydrogenase is inhibited by lycorine (De Tullio et al., Boll. Soc. Ital. Biol. Sper. 70:57-62 (1994); Arrigoni et al., Boll. Soc. Ital. Biol. Sper. 72:37-43 (1996))
Furthermore, the invention provides for a method for increasing the L-ascorbic acid levels in plants, comprising:
a) provision of plants that have been transformed with the sense version of the GLDase gene, and
b) providing the said plants with the precursor L-galactono-xcex3-lactone in order to induce increased L-ascorbic acid synthesis.
According to another aspect thereof the invention provides transgenic plants having in their genome an antisense version of the GLDase gene resulting in a decreased amount of ascorbic acid as compared to non-transgenic plants for use a model system or biosensor for oxidative stress.
The present invention will be elucidated with reference to the non-limitative examples provided below.